The present invention relates generally to compounds and methods for use in inhibiting cadherin-mediated cell adhesion. The invention is more specifically related to modulating agents capable of inhibiting or disrupting interactions between catenin and xcex2-catenin, and to therapeutic methods employing such agents.
The ability of cells to recognize and bind to each other is a fundamental property of multicellular organisms. Such recognition and binding allows for the maintenance of tissue integrity and compartments, and prevents the inappropriate movement of cells and macromolecules between tissues. Cell adhesion also contributes to the natural adhesion of synapses in the body to prevent the remodeling of synapses. The molecules that are responsible for cellular recognition and binding are collectively known as cell adhesion molecules (CAMs).
There are many different families of CAMs, including the immunoglobulin, integrin, selectin and cadherin superfamilies, and each cell type expresses a unique combination of these molecules. Cadherins are a rapidly expanding family of calcium-dependent CAMs (see Munro et al., In. Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes Co. (Austin Tex., 1996)). Examples of the cadherin superfamily include N (neural)-cadherin, E (epithelial)-cadherin, P (placental)-cadherin, and R (retinal-cadherin). These cadherins (termed the classical cadherins, and abbreviated CADs) are integral membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a CAD on the surface of one cell binds to an identical CAD on the surface of another cell), although CADs also appear to be capable of forming heterotypic complexes with one another under certain circumstances and with lower affinity. CADs have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different CADs expressed on different cell types. CADs also regulate the formation of intercellular junctions, and consequently the establishment of physical and permeability barriers between tissue compartments. If cadherin function is abrogated, such junctions between cells do not form.
The structures of the CADs are generally similar. As illustrated in FIG. 1, CADs are composed of five extracellular domains (EC1-EC5), a single hydrophobic domain (TM) that transverses the plasma membrane (PM), and two cytoplasmic domains (CP1 and CP2). The first extracellular domain (EC1) contains the classical cadherin cell adhesion recognition (CAR) sequence, HAV (His-Ala-Val), along with flanking sequences on either side of the CAR sequence that may play a role in conferring specificity.
Inside the cell, the second cytoplasmic domain (CP2) of the classical cadherins interacts with a cytoplasmic protein known as xcex2-catenin (FIG. 1; designated as xcex2) (see Wheelock et al., Current Topics in Membranes 43:169-185, 1996). This protein exists in a complex with another cytoplasmic protein, known as xcex1-catenin (FIG. 1; designated as xcex1). In the absence of this xcex2-catenin/xcex1-catenin complex, the classical cadherins cannot promote cell adhesion. xcex1-catenin also binds to another cytoplasmic protein, known as xcex1-actinin (FIG. 1; designated as ACT), which in turns interacts directly with actin-based microfilaments (FIG. 1; designated as MF) of the cytoskeleton.
xcex2-Catenin is composed of 13 domains, referred to as arm repeats (FIG. 3; see Wheelock et al., Current Topics in Membranes 43:169-185, 1996). The arm repeat closest to the amino terminus of xcex2-catenin (designated as the first arm repeat) is known to contain the xcex1-catenin binding site. The specific amino acids that are directly involved in mediating the interaction between xcex2-catenin and xcex1-catenin have not previously been identified.
Although necessary for a variety of functions in multicellular organisms, CAM function (especially cadherin function) has been implicated in a range of pathological events, including the survival of cancer cells, the migration of cancer cells (metastasis) and the vascularization of tumors (angiogenesis). In such circumstances, it would be advantageous to modulate cadherin function. In order to develop effective therapeutic agents that modulate cadherin function, it is important to further understand the mechanism of cadherin-mediated cell adhesion.
Accordingly, there is a need in the art for improved methods for modulating cadherin function. The present invention fulfills this need and further provides other related advantages.
The present invention provides methods for modulating cadherin-mediated functions. Within certain aspects, the present invention provides modulating agents capable of inhibiting an interaction between xcex1-catenin and xcex2-catenin. In one such aspect, the modulating agent comprises one or more of: (a) the amino acid sequence KHAVV (SEQ ID NO:1); (b) a peptide analogue or peptidomimetic of the amino acid sequence KHAVV (SEQ ID NO:1); or (c) an antibody or antigen-binding fragment thereof that specifically binds to a peptide comprising the amino acid sequence KHAVV (SEQ ID NO:1). Within certain embodiments, the modulating agent comprises the sequence KHAVV (SEQ ID NO:1) within a linear peptide or a cyclic peptide ring. Such modulating agents may, within certain embodiments, comprise a linear or cyclic peptide ranging from 3 to 16 amino acid residues in length.
In another such aspect, a modulating agent comprises: (a) the amino acid sequence HAV; (b) a peptide analogue or peptidomimetic of the amino acid sequence HAV; or (c) an antibody or antigen-binding fragment thereof that specifically binds to a peptide comprising the amino acid sequence HAV; wherein the modulating agent is associated with an internalization moiety. In certain embodiments, the modulating agent comprises a linear or cyclic peptide sequence, which may range from 3 to 16 amino acid residues in length. The internalization moiety may comprise, within certain embodiments, an internalization sequence covalently linked to the modulating agent, a liposome that encapsulates the modulating agent or an antibody or ligand that binds to a cell surface receptor.
Within further embodiments, any of the above modulating agents may be linked to a targeting agent and/or a drug.
Within other aspects, the present invention provides pharmaceutical compositions comprising a cell adhesion modulating agent as described above, in combination with a pharmaceutically acceptable carrier.
The present invention further provides, within other aspects, methods for disrupting an interaction between xcex1-catenin and xcex2-catenin in a cell, comprising contacting a cell with a cell adhesion modulating agent as described above.
Within further related aspects, the present invention provides methods for inhibiting cellular adhesion, comprising contacting a cadherin-expressing cell with a cell adhesion modulating agent as described above.
In other aspects, methods are provided for treating a demyelinating neurological disease in a mammal, comprising administering to a mammal a modulating agent as described above. The modulating agent may be administered, within certain embodiments, by implantation with Schwann cells or by implantation with oligodendrocyte progenitor cells and/or oligodendrocytes.
The present invention further provides, within other aspects, methods for reducing unwanted cellular adhesion in a mammal, comprising administering to a mammal a modulating agent as described above.
Within further aspects, methods are provided for enhancing the delivery of a drug through the skin of a mammal, comprising contacting epithelial cells of a mammal with a drug and a modulating agent as described above, wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of the drug across the epithelial cells. In certain embodiments, the modulating agent passes into the blood stream of the mammal. The modulating agent may be linked to the drug and/or the step of contacting may be performed via a skin patch comprising the modulating agent and the drug.
In other aspects, methods are provided for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal a modulating agent as described above. The modulating agent may be administered to the tumor or may be administered systemically.
Within related aspects, the present invention provides methods for treating cancer in a mammal, comprising administering to a mammal a modulating agent as described above.
The present invention further provides, within other aspects, methods for inhibiting angiogenesis in a mammal, comprising administering to a mammal a modulating agent as described above.
Within further aspects, the present invention provides methods for enhancing drug delivery to the central nervous system of a mammal, comprising administering to a mammal a modulating agent as described above.
Within other aspects, the present invention provides methods for inducing apoptosis in a cadherin-expressing cell, comprising contacting a cadherin-expressing cell with a modulating agent as described above.
Within further aspects, methods are provided for modulating the immune system of a mammal, comprising administering to a mammal a modulating agent as described above.
The present invention further provides, within other aspects, methods for preventing pregnancy in a mammal, comprising administering to a mammal a modulating agent as described above.
Within other aspects, the present invention provides methods for increasing vasopermeability in a mammal, comprising administering to a mammal a modulating agent as described above.
In further aspects, methods are provided for inhibiting synaptic stability in a mammal, comprising administering to a mammal a modulating agent as described above.
Within further aspects, the present invention provides kits for enhancing transdermal drug delivery, comprising: (a) a skin patch; and (b) a modulating agent as described above. The skin patch may be impregnated with the modulating agent and/or may further comprise a drug.
The present invention further provides polynucleotides encoding a modulating agent as described above. Such polynucleotides may be incorporated into a viral vector, such that the modulating agent is generated within a cell infected by the viral vector.
These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually noted for incorporation.